PROJECT SUMMARY The retinoblastoma protein (Rb) pathway is a critical regulator of cell proliferation and a promising target for cancer therapeutics. Rb normally inhibits the transcription program for cell division driven by E2F transcription factors and thus promotes cell cycle arrest in G0/G1. Rb is commonly inactivated by Cyclin-dependent kinase (Cdk) phosphorylation in cancer cells, including by Cdk4/6-CycD complexes. Key unanswered questions in this central cellular pathway include how Cdk4/6 activity is regulated, how specific Rb phosphorylation events mediate E2F activation, why is Rb a more potent tumor suppressive than its close paralogs p107 and p130, and what are the mechanisms of resistance to chemical Cdk4/6 inhibitors. These inhibitors have shown promise in the clinic, but we need to better exploit how they act and what factors influence their response. We will apply our unique combined expertise in biochemical and genetic approaches to answer previously intractable questions about the Cdk4/6-Rb pathway and tumor suppression. Our first goal is to uncover the mechanisms of Cdk4/6-CycD activation in cancer cells. We will use structural, biochemical, and cellular assays to investigate the critical role of the p27 protein in modulating Cdk4/6 activity and the cellular response to Cdk4/6 small molecule inhibitors. Our second goal is to reveal the key molecular changes that occur upon inactivating phosphorylation and the key molecular features that confer tumor suppressor potency to Rb. We will examine how specific Cdk phosphorylation events in Rb lead to its inactivation in cells. We will also explore a small domain in Rb that we hypothesize confers unique tumor suppressive ability compared to p107 and p130. Finally, our third goal is to identify new regulators of the Cdk4/6-Rb pathway using unbiased screening approaches. These new regulators may dictate how we use Cdk inhibitors as therapeutics and innovate new strategies for targeting cancer cell division. These experiments will address fundamental issues in the field of cell-cycle regulation and will transform our understanding of Rb tumor suppressor function, how it is regulated, and how it may be rescued to arrest cancer growth.